The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation

Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator 3 (DIDO3) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here, we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. PHF3 and DIDO exert cooperative and antagonistic effects on the expression of neuronal genes and are both essential for neuronal differentiation. In the absence of PHF3, DIDO3 is upregulated as a compensatory mechanism. In addition to shared gene targets, DIDO specifically regulates genes required for lipid metabolism. Collectively, our work reveals multiple layers of gene expression regulation by the DIDO3 and PHF3 paralogues, which have specific, co-regulatory and redundant functions in transcription.


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April 2023 quantified using the GRCh38/hg38 version of the human transcriptome and the dm6 and mm9 versions of the Drosophila (RNA-seq) and mouse (3'end mRNA-seq) spike-in transcriptome (downloaded from the ENSEMBL database, doi:10.1093/nar/gkx1098)using SALMON (version 1.9.0,DOI: 10.1038/nmeth.4197)with default parameters.For visualization purposes, the data was mapped to the GRCh38/hg38, dm6 and mm9 versions of the human, drosophila and mouse genomes using STAR (version 2.7.10a), with the following parameters: --limitOutSJcollapsed 20000000 --limitIObufferSize=1500000000 --outFilterMultimapNmax 10 --seedPerWindowNmax 5.The quantified data was processed using tximport (DOI: 10.12688/f1000research.7563.2),and the differential expression analysis was done using DESeq2 (version 1.38.1, DOI: 10.1186/s13059-014-0550-8).Proliferation assay data was analysed and plotted using GraphPad Prism (9.1.1).FACS cell cycle analysis data were analysed in FlowJo (version 10.8.1).Raw mass spectrometry data from pS5 IP PHF3 samples and FLAG IP with DIDO samples were processed using the MaxQuant software package (version 1.6.0.16) and the Uniprot human reference proteome (July 2018, www.uniprot.org)as well as a database of most common contaminants.Downstream data analysis was performed using the LFQ values in Perseus (version 1.6.2.3).To determine differentially enriched proteins we used the LIMMA package in R (version 3.5.1.)and applied the Benjamini-Hochberg correction for multiple testing to generate adjusted p-values.For pS5 IP DIDO samples, MS raw data split for each CV using FreeStyle 1.7 (Thermo Fisher) were analysed using the MaxQuant software package (version 2.1.0.0) with the Uniprot human reference proteome (version 2022.01, www.uniprot.org),as well as a database of most common contaminants.MaxQuant output tables were further processed in R 4.2.0 (https://www.R-project.org)using Cassiopeia_LFQ (https://github.com/moritzmadern/Cassiopeia_LFQ).To determine differentially enriched proteins we used the LIMMA package in R (version 3.5.1.)and applied the Benjamini-Hochberg correction for multiple testing to generate adjusted p-values.Condensate size in LLPS assays was analysed using Fiji (ImageJ 1.53c).
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RT qPCR was performed in 3-5 biological replicates and each individual sample was measured in technical triplicates.This sample size was chosen because it allows for statistical analysis and detection and removal of potential outliers.Since we were in all cases able to replicate our results, we deemed the sample size sufficient.Sample sizes and sequencing depth for NGS experiments were chosen based on the standards recommended by the ENCODE Consortium and on previously published similar studies.ENCODE recommends two or more biological replicates for RNA-seq.We chose to perform RNA-seq and 3'polyA mRNA-seq in 3 biological replicates because this allows for detection and removal of potential outliers.All attempts at replication of NGS data were successful, therefore we consider the sample size sufficient.Proliferation assays, FACS cell cycle analysis, Co-IPs followed by mass spectrometry, Co-IPs followed by Western Blotting and Airyscan colocalization analysis experiments were performed in triplicates (with multiple cells per replicate being analysed for Airyscan colocalization analysis) because this allows for statistical analysis and detection and removal of potential outliers.Since we were in all cases able to replicate our results in all three replicates, we deemed the sample size sufficient.Sucrose gradient ultracentrifugation to show DIDO-PHF3 complex formation was performed once, but complex formation in intact cells was further assessed by microscopy-based colocalization assays.
For LLPS assays at least 250 droplets per condition were imaged, the sample size was chosen based on previously published similar studies.
Neuronal differentiation experiments were performed in 4 biological replicates to allow for detection and removal of potential outliers.Since the results were replicated in all replicates we consider the sample size sufficient.Genotyping PCRs, Western blots and immunofluorescence during cell line generation were performed once, since they were meant to confirm the genotype, protein expression and protein localization and not to generate experimental data.
Data exclusions No data were excluded from analysis.

Replication
All attempts at replication were successful.RT qPCR was performed in 3-5 biological replicates with similar results (the precise number of biological replicate for each experiment is provided in the corresponding figure legend), each individual sample was measured in technical triplicates.
Mass spectrometry experiments were performed in three biological replicates, results were successfully replicated.Co-IP experiments followed by Western blotting were performed to confirm results from mass spectrometry analysis.These experiments were performed once, they confirmed the interactors identified by mass spectrometry.Sucrose gradient ultracentrifugation to show DIDO-PHF3 complex formation was performed once.We did not replicate this experiment because we also assessed complex formation in intact cells using a different, independent method through microscopy-based colocalization assays.Colocalization assays were performed in biological triplicates with multiple cells imaged per replicate.Both methods independently confirmed complex formation.LLPS assays were performed in two independent replicates with at least 250 droplets imaged per replicate (precise number of droplets imaged is provided in the figure legend).The results were successfully replicated.RNA-seq experiments were performed in three biological replicates and were successfully replicated.
Immunofluorescence analysis was performed in two biological replicates with multiple cells imaged per replicate.The results were successfully replicated.Proliferation assays were performed in three biological replicates with similar results.FACS cell cycle analysis was performed in three biological replicates with similar results.Neuronal differentiation assays were performed in four biological replicates with similar results.Experiments meant to confirm successful CRISPR/Cas9 editing rather than generate experimental data (genotyping PCRs, Western Blots, IF) were not replicated.
Randomization Randomization was not performed for this study.Samples (cell lines) were allocated to experimental groups based on genotype and paired with a control, i.e.KO and domain deletion cell lines were compared to a control WT cell line or cells transfected with FLAG-or HA-constructs were compared to empty vector transfected control cells.All cell lines were grown under the same conditions and experiments were performed under identical conditions.

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The investigators were not blinded in this study since they were involved in the planning, execution and analysis of the experiments.Withinexperiment sample groups and the respective controls were prepared, processed and analysed at the same time and under identical conditions to eliminate any bias, so prior knowledge had no impact on data output.No subjective process was involved in the analysis of the data.Analysis of NGS-and mass spectrometry data was carried out bioinformatically applying the same parameters to all samples without need for investigator blinding.

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Mouse anti-GFAP (Sigma G3893): Manufacturer's statement: The antibody reacts specifically with GFAP in immunoblotting assays and labels astrocytes, Bergmann glia cells and chondrocytes of elastic cartilage in immunohistochemical staining.The antibody reacts with glial specific antigen in frozen or alcohol-fixed tissue sections.Images demonstrating immunofluorescence staining are provided on the manufacturer's website.For FACS during cell line generation, singlet population was defined by forward vs. side scatter (FSC vs. SSC) gating.Within the singlet population, populations were gated based of their GFP or mScarlet fluorescence (depending on the editing approach): GFP-/mScarlet-(negative), GFP+ high (exogenous GFP-expression after transfection with pX458 plasmids), GFP+ low (endogenous GFP-expression, generation of endogenously GFP-tagged cell lines) and mScarlet+ low (endogenous mScarlet expression, generation of endogenously mScarlet-tagged cell lines).
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For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdfnature portfolio | reporting summary If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.DIDO isoforms in HepG2 cell lysate is provided on the manufacturer's website.Mouse anti-Human DIDO1 (R&D Systems MAB6947): Manufacturer's statement on specificity testing by Western Blot: Detection of Human DIDO1 by Western Blot.Western blot shows lysates of Raji human Burkitt's lymphoma cell line.Gels were loaded with 30 μg of whole cell lysate (WCL), 20 μg of cytoplasmic (Cyto), and 10 μg of nuclear (Nuc) extracts.PVDF membrane was probed with 2 μg/ mL of Mouse Anti-Human DIDO1 Monoclonal Antibody (Catalog # MAB6947) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007).A specific band was detected for DIDO1 at approximately 280 kDa (as indicated).Mouse anti-FLAG cl.M2 (Sigma F1804): Statement from the manufacturer: For highly sensitive and specific detection of FLAG fusion proteins by immunoblotting, immunoprecipitation (IP), immunohistochemisty, immunofluorescence and immunocyotchemistry.Optimized for single banded detection of FLAG fusion proteins in mammalian, plant, and bacterial expression systems.Image of immunofluorescence detection of a FLAG-tagged protein is provided on the manufacturer's website.Validated for IF in human cells in DOI 10.1186/s12985-016-0610-7, where FLAG-tagged proteins expressed in HeLa cells were detected by IF using mock transfected cells as a negative control.Anti-GFP antibody (ab290) is a highly versatile antibody that gives a stronger signal than other anti-GFP antibodies available.OnWestern blot the antibody detects the GFP fraction from cell extracts expressing recombinant GFP fusion proteins and has also been shown to be useful on mouse sections fixed with formalin.In Immunocytochemistry, the antibody gives a very good signal on recombinant YES-GFP chimeras expressed in COS cells(McCabe et al.  1999 and figure below).It is routinely used in Immunoprecipitation (IP) and IP-Western protocols and has been used successfully in HRP Immunohistochemistry at 1:200 on whole-mount mouse embryos.Mouse anti-GFP (Roche 11814460001): Manufacturer's statement on quality control: Anti-GFP is tested for functionality and purity relative to a reference standard to confirm the quality of each new reagent preparation.Validated for Immunofluorescence in DOI:10.1093/cvr/cvs277and https://doi.org/10.1242/dev.043703.Rabbit anti-H3K9ac (Sigma 07-352): Manufacturer's statement: Broad species-cross reactivity is expected.Recognizes acetyl-histone H3 (Lys9), Mr 17 kDa.An additional unknown protein was detected at Mr 23 kDa.Images demonstrating detection of H3K9ac by immunofluorescence are provided on the manufacturer's website.Detection of H3K9ac in human cells was demonstrated e.g. in doi: 10.1111/cas.12717.Rabbit anti-H3K9me3 (abcam ab8898): Manufacturer's statement: Histone H3 (tri methyl K9) antibody (ab8898) is specific for Histone H3 tri methyl Lysine 9. Shows slight cross-reactivity with tri methyl K27, which shares a similar epitope.Does not react with mono or di methylated K9.Validated for Immunofluorescence by the manufacturer in Mouse 3T3MEF, Indian muntjac fibroblast cells, HeLa cells and Mouse Embryonic Stem cells.Immunofluorescence images for HeLa cells are provided on the manufacturer's website.
Rabbit anti-GFP (polyclonal), abcam ab290 Mouse anti-GFP cl.13.1 and cl.7.1 (mixture of two monoclonal antibodies), Roche 11814460001 Rabbit anti-H3K9ac (polyclonal), Sigma 07-352 Rabbit anti-H3K9me3 (polyclonal), abcam ab8898 Rabbit anti-fibrillarin cl.C13C3, Cell Signaling 2639 Mouse anti-HA.11cl.16B12,Covance901513Rabbit anti-HCFC1 (polyclonal), Cell Signaling 69690 Rabbit anti-histone macro H2A.1 (polyclonal), abcam ab37264 Rabbit anti-histone macro H2A1.2 (polyclonal), Cell Signaling 4827 Rabbit anti-H2AZ (polyclonal), Cell Signaling 2718 Rabbit anti-gH2AX (polyclonal), Bethyl A300-081A Rabbit anti-PAF1 (polyclonal), abcam ab20662 Mouse anti-PARP cl.C2-10, Trevigen 4338-MC-50 Mouse anti-PARP2 cl.4G8, Enzo ALXmanufacturer, the antibody detects DEK in wild type 293T, but not DEK KO 293T cells.Rabbit anti-DIDO1 (Atlas antibodies HPA049904): Manufacturer's statement on validation: Validated in Western blot using relevant lysates.An image of Western Blot detection in the human cell line RT-4 using this antibody is shown on the manufacturer's website.Target specificity was validated in this study and our previous study (https://doi.org/10.1038/s41467-023-35853-1),where it was used to detect loss of DIDO protein after CRISPR/Cas9 knock-out by Western Blotting and IF.Rabbit anti-Pan DIDO (Millipore ABN1367): Manufacturer's statement on specificity: This polyclonal antibody detects an N-terminal epitope present in all four spliced isoforms of human DIDO1 reported by UniProt (Q9BTC0).Image showing Western Blot detection of nature portfolio | reporting summary Rabbit anti-fibrillarin (C13C3) (Cell Signaling 2639): Manufacturer's statement: Fibrillarin (C13C3) Rabbit mAb detects endogenous levels of total fibrillarin protein.Immunofluorescence images for mouse tissues and HeLa cells are provided on the manufacturer's website.Mouse anti-HA.11cl.16B12(Covance901513):Manufacturer's statement on quality control: Each lot of this antibody is quality control tested by Western blotting.Validated by the manufacturer for Western Blotting and IF (images of Western Blot and IFdetection in cell lines transfected with and HA-construct using non-transfected cells as a control are provided on the manufacturer's website) and in this study (Fig.7e,f; detection of HA-constructs expressed in HEK293T cells using empty vector transfected cells as a negative control).Rabbit anti-HCFC1 (Cell Signaling 69690): Manufacturer's statement on specificity: HCFC1 Antibody (Amino-terminal Antigen) recognizes endogenous levels of total HCFC1 protein.This antibody also recognizes amino-terminal fragments (HCFC1-N) resulting from O-GlcNAc transferase (OGT) cleavage.Western Blot image of HCFC1 detection in lysates from human cells is provided on the manufacturer's website.Rabbit anti-histone macro H2A.1 (abcam ab37264): Manufacturer's statement on specificity: ab37264 recognises the three known isoforms of mH2A1 including mH2A1.2 (longest isoform) and the mH2A1.1 (shortest isoform).Images for Western Blot detection in human cell lines HeLa and HepG2 are provided on the manufacturer's website.Target specificity validated by the manufacturer using HAP1 WT and KO cells, Western blot image provided on the manufacturer's website.Rabbit anti-histone macro H2A1.2 (Cell Signaling 4827): Manufacturer's statement on specificity: MacroH2A1.2Antibody detects endogenous levels of the core histone MacroH2A1.2protein (MacroH2A1, isoform 2).The antibody does not cross-react with MacroH2A1.1 (MacroH2A1, isoform 1), MacroH2A2 or histone H2A.Images showing Western Blot detection in human cell lysates (HeLa, H-4-II-E) and MacroH2A1.2transfected cells are provided on the manufacturer's website.Rabbit anti-H2AZ (Cell Signaling, 2718): Manufacturer's statement on specificity: Histone H2A.Z Antibody detects endogenous levels of histone H2A.Z protein.The antibody does not cross-react with other histone proteins, including histone H2A.
April 2023tissues and through the recent efforts of the Human Cell Atlas project, many have been characterized by immunofluorescence to map the human proteome not only at the tissue level but now at the subcellular level.These images and the collection of this vast data set can be viewed on the Human Protein Atlas (HPA) site by clicking on the Image Gallery link.Rat phospho-CTD antibodies cl.3E8, cl.3E10 and cl.4E12 were validated in DOI: 10.1126/science.1145977Mouse anti-Pol II 4H8 (Cell Signaling) -Manufacturer's statement on Specificity: 'Rpb1 CTD (4H8) Antibody detects endogenous levels of total Rpb1 protein (both phosphorylated and unphosphorylated forms).'Images confirming Western Blot detection in human cells are provided on the manufacturer's website.Mouse anti-Pol II clone F-12 (Santa Cruz sc-55492): used in >30 publications and validated in our previous study (https:// doi.org/10.1038/s41467-021-26360-2)using purified Pol II complex in WB and in ChIP.Mouse anti-SPT5 (Becton Dickinson 611107) -Manufacturer's statement: 'This antibody is routinely tested by western blot analysis.'Rabbit anti-SPT6 (Novus Biologicals NB100-2582): Validated by the manufacturer using siRNA knockdown to confirm the specificity of Spt6 antibody in C2C12 myoblasts.The antibody was validated for Western Blotting in human cells in Lu et al. 2020 (https:// doi.org/10.1172/JCI138577),where it was used to confirm SPT6 depletion in MDA-MB-231 cells transfected with shRNAs targeting SPT6 by Western Blotting.
PARP-2.Image of Western Blot detection in HeLa cells is provided on the manufacturer's website.Rabbit anti-PHF3 (Atlas antibodies HPA025763): Validated in our previous study (https://doi.org/10.1038/s41467-021-26360-2),where it was used to detect loss of PHF3 protein after CRISPR/Cas9 knock-out by Western Blotting.Statement from the manufacturer: All Prestige Antibodies Powered by Atlas Antibodies are developed and validated by the Human Protein Atlas (HPA) project and as a result, are supported by the most extensive characterization in the industry.The Human Protein Atlas project can be subdivided into three efforts: Human Tissue Atlas, Cancer Atlas, and Human Cell Atlas.The antibodies that have been generated in support of the Tissue and Cancer Atlas projects have been tested by immunohistochemistry against hundreds of normal and disease nature portfolio | reporting summary Rat Monoclonal Antibody (Product # MA1-80017).The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2μg/mL Rat primary antibody for 3 hours at room temperature.Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate (Product # A-11007) was used at a concentration of 4μg/mL in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red).Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938).F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379), 1:300) (Panel c: green).Panel d represents the composite image.No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e).The images were captured at 60X magnification.Goat anti-rat AF647 (abcam ab150167): Manufacturer's statement: By immunoelectrophoresis and ELISA this antibody reacts specifically with rat IgG and with light chain common to other rat immunogobulins.No antibody was detected against nonimmunoglobulin serum proteins.Less than 1% cross reactivity to bovine, chicken, human, mouse, rabbit and sheep IgG was detected.This antibody may cross react with IgG from other species.Tested for immunofluorescence by the manufacturer.Images for HeLa cells are provided on the manufacturer's website.Goat anti-rabbit AF647 (ThermoFisher A21244): Manufacturer's statement on antibody testing for IF in human cells: Immunofluorescence analysis of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 647 conjugate was performed using HeLa cells stained with alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-16891).The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2 μg/mL primary antibody for 3 hours at room temperature.Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 647 conjugate (Product # A-21244) was used at a concentration of 4 μg/mL in phosphate buffered saline containing 0.2% BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red).Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938).F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379), 1:300) (Panel c: green).Panel d represents the composite image.No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e).The images were captured at 60X magnification.Eukaryotic cell linesPolicy information about cell lines and Sex and Gender in Research Cell line source(s) HEK293T (CRL-3216), MEFs (CRL-2991) and Drosophila S2 (CRL-1963) were obtained from ATCC.
2% BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red).Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938).F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379), 1:300) (Panel c: green).Panel d represents the composite image.No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e).The images were captured at 60X magnification.Goat anti-rat AF594 (ThermoFisher A11007): Manufacturer's statement on antibody testing in IF in human cells: Immunofluorescence analysis of Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate was performed using A549 nature portfolio | reporting summary